PROJECT SUMMARY/ABSTRACT Although it has long been thought that an AUG start codon universally marks the beginning of an open reading frame, recent advances in ribosome footprint mapping have revealed that ribosomes initiate translation at non- AUG codons (e.g., CUG or GUG) at an astonishing frequency. Canonical AUG start codons are recognized by the initiator tRNAi within the scanning pre-initiation complex, but it is still largely unclear how non-AUG start Met codons are recognized or regulated. Considering that aberrant forms of non-AUG translation have been shown to cause multiple neurodegenerative diseases and contribute to cancer malignancy, a clearer understanding of how non-AUG translation is controlled on the molecular level is greatly needed. Using plasmids that express the same reporter protein but from different start codons, I surprisingly found that a reporter expressed from a CUG or GUG start codon was not inhibited by well-characterized translation inhibitors and, in fact, increased over time. This is in stark contrast to the expected inhibition of translation I observed when the reporter initiated from a canonical AUG. These data strongly indicate that ribosomes translating proteins from non-AUG start codons are fundamentally different from ribosomes that start at AUG codons. During the mentored phase, I will gain new training in biochemistry and bioinformatics to address how translation at different start codons is uniquely regulated by both the ribosome and sequence elements within mRNAs. In Aim 1, I will affinity purify translating reporter mRNAs that initiate from an AUG or GUG start codon and determine the composition of the bound ribosomes, as well as identify other associated proteins that may confer resistance to translation inhibitors. These experiments will reveal important insights into how ribosomes translating distinct open reading frames can be different and be controlled by separate regulatory pathways. In Aim 2, I will use a candidate gene approach along with genome-wide ribosome profiling to identify sequences that regulate non- AUG translation from endogenous genes. By identifying and characterizing the key regulatory sequences, this aim should allow us to predict and validate which genes are translated by separate ribosome classes within cells. In Aim 3, I will take full advantage of this training and determine in my own laboratory how non-AUG translation is regulated when protein synthesis is naturally attenuated during cell stress. In particular, we will use ribosome profiling to identify non-AUG open reading frames that are regulated during cell stress and characterize how these events impact the stress response and cell survival. In the short term, the proposed new training (Aims 1 & 2) by my expert mentors and collaborators, along with complementary formal coursework, will provide a foundation on which I can continue to reveal in my independent laboratory how non- AUG translation is regulated and controls human biology (Aim 3). The excellent training environment in the Wilusz & Dreyfuss laboratories and at Penn will greatly facilitate not only the mentored research, but also endow me with the necessary professional skills to transition to an independent faculty position.